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Noncoding RNAs in cardiovascular disease – potential as biomarkers and more
Ali Bierly, [email protected] 19, 2016
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Agenda
1. Introduction to cardiovascular system and diseases
2. Introduction to noncoding RNAs (microRNA and lncRNA)
3. Noncoding RNAs in cardiovascular diseases – what’s known, and new research
4. Tools for total RNA research (mRNA, miRNA and lncRNA)
5. Additional resources
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Cardiovascular diseases
Diseases of the heart and blood vessels
Coronary artery
disease
High blood pressure
Congestive heart failure
Cardiac arrestArrythmia
Peripheral artery
disease
Stroke
Top cause of death globally – includesdiseases of the heart and blood vessels
Behavioral risk factors: Tobacco use Diet/obesity Physical inactivity Excessive alcohol use
Early detection and management is key to successful control*
* Source: WHO Cardiovascular diseases (CVDs) fact sheet
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The cardiovascular system
Image CC BY-SA 2.1 JP http://creativecommons.org/licenses/by-sa/2.1/jp/
Primary responsibilities: Blood circulation, leading to:
Nutrient transport, such as amino acids Oxygen transport CO2 transport Hormone circulation Blood cell circulation
Primary organs/vessels involved: Heart (pumps blood to body and lungs) Lungs (pulmonary circulation, oxygenates blood) Arteries (deliver oxygenated blood) Veins (return blood to the heart) Capillaries (connect arteries and veins)
Blood component of the circulatory system, which also includes the lymphatic system
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Major cardiovascular diseases
Vascular diseases
Coronary artery disease (can lead to myocardial infarction)Peripheral arterial diseaseCerebrovascular disease (including stroke)Renal artery stenosis
Heart diseases
Cardiomyopathy – diminished ability of heart muscle to contractHypertrophic cardiomyopathy – the above, due to thickening of myocardium Hypertensive heart disease – complications of high blood pressure; left ventricular hypertrophy, atherosclerosis, congestive heart failure, atrial fibrillationHeart failure – heart can’t pump enough anymore, can result from a number of CVDsValvular heart disease
Question: Can noncoding RNA biomarkers help us detect CVD early?
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Noncoding RNAs – microRNA overview
microRNA (miRNA) Naturally occurring, ~22-nt noncoding RNAs Mediate post-transcriptional gene regulation Circulating miRNAs are detectable in serum and plasma – good potential biomarkers Detectable by qPCR, sequencing or microarray or a discovery-verification combination of
sequencing followed by qPCR Involved in many biological pathways, including apoptosis, cell differentiation and
development and immunity, as well as diseases from cancer to cardiovascular to diabetes
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microRNA biogenesis
Noncoding RNAs – microRNA overview
Transcribed by RNA polymerase II as long primary transcripts (pri-miRNAs), which may contain more than one miRNA
In the nucleus, pri-miRNAs are processed to hairpin-like pre-miRNAs by RNAse III-like enzyme Drosha
Pre-miRNAs are then exported to the cytosol by Exportin 5
In the cytosol, RNAse III-like Dicer processes pre-miRNAs into mature miRNAs
Mature miRNAs are incorporated into RISC
miRNAs with high homology to target mRNAs lead to mRNA cleavage
miRNAs with imperfect base pairing to target mRNAs lead to translational repression and/or mRNA degradation
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Long noncoding RNA (lncRNA) Noncoding transcripts >200 nt (some may code for “micropeptides”)
Not strongly conserved across species in general, but some strongly conserved elements
Involved in regulating gene transcription, post-transcriptional regulation and epigenetic regulation
Noncoding RNAs – lncRNA overview
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lncRNAs are a novel class of RNAs larger than 200 nucleotides
Noncoding RNAs – lncRNA overview
Martin, L., Chang, H.Y. (2012) Uncovering the role of genomic "dark matter" in human disease. J Clin. Invest. 122, 1589
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What’s currently known about ncRNA in CVD research?
Acute coronary syndromes Coronary artery disease Heart failure diagnosis
Upregulated
miR-1, miR-21, miR-30a, miR-30c, miR-34a, miR-122, miR-126, miR-133a/b, miR-134, miR-145, miR-146a, miR-155, miR-186, miR-195, miR-198, miR-199, miR-208, miR-208b, miR-223, miR-320a, miR-328, miR-370, miR-423-5p, miR-433, miR-485-3p, miR499
Upregulated
miR-21, miR-25, miR-92a, miR-106b, miR-122, miR-133a, miR-135a, miR-140-3p, miR-146a, miR-155, miR-182, miR-186, miR-208b, miR-370, miR-451, miR-490-3p
Upregulated
miR-18b*, miR-21, miR22, miR-29b, miR-30a, miR-92b, miR-122, miR-129-5p, miR-133a, miR-142-3p, miR-200b, miR-210, miR-320a, miR-423-5p, miR-499, miR-519e*, miR-520d-5p, miR-622, miR-675, miR-1254, HS_202.1
Downregulated
let-7b, miR-29a, miR-122, miR-125b, miR-126, miR-155, miR-223, miR-320b, miR-375, miR-663b, miR1291
Downregulated
miR-21, miR-29a, miR-31, miR-125b, miR-147, miR-181a, miR-214, miR-320b
Downregulated
miR-30b, miR-103, miR-107, miR-125b, miR-126, miR-139, miR-142-3p, miR-142-5p, miR-342-3p, miR-497
Information from Romaine, S.P.R. et al. (2015) microRNAs in cardiovascular disease: an introduction for clinicians. Heart doi:10.1136/heartjnl-2013-305402
microRNAs with differential expression in cardiovascular diseases
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* Information from Uchida, S. and Dimmeler, S. (2015) Long noncoding RNAs in cardiovascular diseases. Circ. Res. 116, 737.
** Information from Kataoka, M. and Wang, D.-Z. (2014) Non-coding RNAs I including miRNAs and lncRNAs in cardiovascular biology and disease. Cells 3, 883.
What’s currently known about ncRNA in CVD research?
lncRNAs that have been associated with cardiovascular diseases
Acute myocardial infarction: HIF-1 AS*, MALAT1*, KCNQ1OT1*, MIAT**, ANRIL NR_003529*
Cardiac hypertrophy: CHRF (induces hypertrophy by acting as a sponge formiR-489, de-repressing its target MyD88)*, Mhrt (downregulated after pressure overload, protective of heart function when overexpressed)*
Coronary artery disease susceptibility: ANRIL**
Many more are being investigated by RNA-seq and greater characterization is yet to come.
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Recent advances in noncoding RNA biomarkers for CVDs
microRNAs in circulating HDL help predict vulnerability of coronary artery disease patients
Niculescu, L.S. et al. (2015) miR-486 and miR-92a Identified in Circulating HDL Discriminate between Stable and Vulnerable Coronary Artery Disease Patients.
PLoS One 10, e0140958.
Aim: Determine if there’s any relationship between the microRNAs associated with circulating lipoproteins and stability or vulnerability in CAD patients
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What is coronary artery disease?
Cause: Buildup of plaque in the arteries (atherosclerosis), restricting flow of oxygen-enriched blood to the heart by narrowing the arteries. Plaque breaking up can result in blood clot formation on top of the ruptured plaque, blocking blood flow.
Possible consequences: Angina (chest pain)
Myocardial infarction (heart attack, death of heart muscle due to lack of blood flow)
Arrhythmias
Heart failure
What is a stable vs vulnerable plaque?
A vulnerable plaque is at risk for rupturing, causing blood clots and driving patients toward heart attack. A key goal in the CAD field is to find an early detection method for vulnerable plaques / vulnerable patients in order to help prevent coronary events.
Image: Blausen.com staff. "Blausen gallery 2014". Wikiversity Journal of Medicine. DOI:10.15347/wjm/2014.010. ISSN 20018762. Used under Creative Commons Attribution 3.0 Unported license.
Question: Can microRNA biomarkers help identify vulnerable CAD patients?
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Niculescu et al.’s approach: qPCR and statistical analysis
Four groups of subjects: stable angina (SA), unstable angina (UA), 1 month post-myocardial infarction (MI) and healthy controls
Isolated serum for biochemical and microRNA analysis, and generated 3 independent pools for each group of subjects.
Profiled serum parameters (cholesterol, triglycerides, fasting glucose, etc.) Isolated/characterized serum lipoproteins (IDL, LDL, HDL, HDL2, HDL3) by
ultracentrifugation
Isolated and analyzed miRNAs from sera and lipoprotein fractions
Used miRNeasy Serum/Plasma Kit for isolation Screened serum miRNAs in a pool of sera from 8 random individuals per group with
Human CVD miScript miRNA PCR Array Used individual miRNA TaqMan assays for 7 microRNAs
in all 111 (95 CAD, 16 control) subjects Used binary logistic regression model to determine predictive
value of the microRNAs for vulnerable CAD patients
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Findings and relevance – miRNA biomarkers in CAD
From the array, they found 48 upregulated miRNAs in serum of SA, 38 in UA and 38 in MI – MI group’s microRNAs were considered top-rank
The highest ranked were miR-486, miR-92a and miR-122, and the group analyzed them with individual qPCR assays as well as 3 others due to previous literature (miR-146a, miR-125a and miR-33a)
No significance in differences between SA and UA serum in the larger cohort for these microRNAs; small & significant increase for miR-122, miR-146a and miR-125a in MI vs SA
The 6 selected microRNAs were mostly associated with HDL, varying by subfraction
HDL from CAD patients showed highest levels of miR-486 and miR-92a (predominantly associated with HDL2 and HDL3, respectively)
miR-486 was much higher in HDL2 from UA and MI compared to SA miR-92a in HDL3 was higher in UA and MI compared to SA
miR-486 and miR-92a associated with HDL can distinguish between vulnerable and stable CAD patients – may warrant further study along with a combination of these miRNAs with apolipoprotein levels, PON1 activity and HDL/LDL ratio (data not discussed here)
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Recent advances in noncoding RNA biomarkers for CVDs
microRNA biomarkers identify diffuse myocardial fibrosis
Fang, L. et al. (2015) Circulating microRNAs as biomarkers for diffuse myocardial fibrosis in patients with hypertrophic cardiomyopathy. J. Transl. Med. 13, PMID: 26404540
Aim: To find circulating biomarkers for myocardial fibrosis to alleviate the problems of traditional cardiac magnetic resonance (CMR) imaging techniques for diagnosis.
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Diffuse myocardial fibrosis and hypertrophic cardiomyopathy
Hypertrophic cardiomyopathy (HCM): Thickening of the myocardium that damages cardiac muscle function and can lead to heart
failure or unexpected cardiac death.
Usually asymptomatic
Genetic component – can be inherited (often a mutation in the beta myosin heavy chain or the cardiac myosin binding protein C gene) or de novo mutation in beta myosin heavy chain gene
Diffuse myocardial fibrosis: Early feature of HCM
Associated with poor prognosis
Difficult to diagnose
Image: Blausen.com staff. "Blausen gallery 2014". Wikiversity Journal of Medicine. DOI:10.15347/wjm/2014.010. ISSN 20018762. Used under Creative Commons Attribution 3.0 Unported license.
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Fang et al.’s approach
Used cardiac magnetic resonance (CMR) imaging and postcontrast T1 mapping time to identify diffuse myocardial fibrosis in HCM patients
Collected blood samples from the same patients prior to CMR and isolated RNA from plasma using the miRNeasy Mini Kit.
microRNA was surveyed with the miScript Serum & Plasma miRNA PCR Array
Individual miRNA assays were used to follow up the array results on dysregulated miRNAs
Receiver operating characteristic (ROC) curve was used to determine how individual miRNAs could diagnose fibrosis, and a logistic regression model was used to determine probabilities.
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Findings and relevance: biomarkers for diffuse myocardial fibrosis
Identified 14 miRNAs that were increased in patients with diffuse myocardial fibrosis compared to healthy controls in the initial screen of 8 HCM patients and 4 controls
12 of these were confirmed by individual qPCR assays in a larger cohort of 55 HCM patients
AUC for all 14 miRNAs, the 12 validated miRNAs from the screen plus miR-29a-3p and miR-133-3p, was 0.87 for prediction of diffuse myocardial fibrosis
The logistic regression model with backward stepwise method narrowed this down to 8 miRNAs, still with an AUC of 0.87
miRNAs identified as potential biomarkers for myocardial fibrosis
miR-18a-5p miR-30d-5p miR-21-5p miR-193-5p
miR-10b-5p miR-296-5p miR-29a-3p miR-15a-5p
This microRNA signature might someday be developed into an alternative diagnostic option that is less expensive and more available than CMR, with no contraindications for renal dysfunction or implanted cardiac devices.
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Recent advances in noncoding RNA biomarkers for CVDs
High plasma expression of microRNAs in coronary artery disease
Zhou, J. et al. (2016) miRNA 206 and miRNA 574-5p are highly expression [sic] in coronary artery disease. Biosci. Rep. 36, e00295
Aim: Identify key microRNAs in CAD that could be turned into diagnostic biomarkers in the future.
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Zhou et al.’s approach:
Isolated blood from CAD patients and healthy controls (defined as individuals with no coronary stenosis or atherosclerotic vascular disease)
Initial screening of microRNAs was done via microarray
Individual microRNA assays (miScript Primer Assays) were used to follow up by qPCR
Used statistical analysis to determine the strength of the microRNAs’ predictive ability for CAD
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Findings and relevance: high miRNA expression in CAD
Identified 33 microRNAs differentially expressed in CAD patient plasma (3 samples)
2 microRNAs were selected for analysis in a larger sample group, miR-206 and miR-574p – they were upregulated very strongly compared to controls (8.74-fold and 29.53-fold) and their targets were possibly related to CAD development
miR-206 and miR-574p were confirmed as consistently upregulated in CAD via qPCR assay in 67 CAD patients and 67 healthy controls
ROC curve analysis showed a 0.607 AUC value for miR-206 and a 0.696 value for miR-574-5p
These 2 microRNAs are possibly promising for development of early biomarkers for CAD
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Technologies for total RNA discovery – Isolation
Total RNA isolation – miRNeasy Kits
FFPE KitMicro KitMini KitSerum/Plasma Kit
Purify total RNA 18 nt and up, including mRNA, miRNA and lncRNA
Can also purify separate miRNA-enriched fraction and total RNA >200 nt fractions (Mini/Micro kits)
Products are suitable for quantitative RT-PCR, Northern blot and microarray analysis
Automatable on QIAcube
Use with up to 200 µl serum or plasma
Internal normalization controlavailable
Visit webpage for more info
Animal cells/tissues, including difficult-to-lyse tissues
Visit webpage for more info
Works with small amounts of cultured cells (1 x 106 cells)
Works with small amounts of animal/human tissue (≤5 mg)
Visit webpage for more info
Lysis buffer efficiently releases
RNA while avoiding degradation
Subsequent incubation at 80°Creverses formalin crosslinking
Visit webpage for more info
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Technologies for total RNA discovery – mRNA expression
RT2 Profiler PCR Arrays and Assays Available for 13 species
Lab-verified assays
Multiple arrays for CVD-related pathways, with gene lists selected by our experts:
Endothelial cell biology Atherosclerosis Lipoprotein signaling & cholesterol metabolism Angiogenesis VEGF signaling Cardiotoxicity Hypertension View the full list
Preamplification Optional for small samples – RT2 PreAMP cDNA Synthesis
Kit and Primer Mixes
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Profile 84 different genes using one array
Appropriate controls for
data normalization sample quality reaction performance
Lab-verified assays for guaranteed performance
Gene expression profiling – RT2 Profiler PCR Arrays & Assays
Technologies for total RNA discovery – mRNA expression
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Technologies for total RNA discovery – microRNA expression
microRNA expression profiling and functional analysis
qPCR arrays and assays
miScript microRNA PCR Arrays and Primer Assays
CVD-related arrays: Cardiovascular Disease
Apoptosis
Cell Differentiation & Development
Serum and Plasma
miFinder
Full miRNome arrays: Through miRBase V21 for human and
mouse
Leading coverage for dog, rat, rhesus macaque and cow
Functional analysis miScript miRNA Mimics
miScript miRNA Inhibitors
miScript miRNA Target Protectors
qPCR reagents and preamplification miScript PreAMP PCR Kit and Primer
Mixes (optional step for small samples)
miScript II RT Kit
miScript SYBR Green PCR Kit
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Technologies for total RNA discovery – microRNA expression
Profile 84 different miRNAs using one array
Appropriate controls for
data normalization sample quality reaction performance
Lab-verified assays for guaranteed performance
miScript miRNA PCR Arrays
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microRNA list for the Human Cardiovascular Disease miScript array
Technologies for total RNA discovery – microRNA expression
Myocardial Infarction:Up-Regulated: let-7e-5p, miR-122-5p, miR-126-5p, miR-133b, miR-145-5p, miR-146a-5p, miR-15b-5p, miR-208a, miR-208b, miR-223-3p, miR-320a, miR-499a-5p.Down-Regulated: miR-1, miR-107, miR-130a-3p miR-133a, miR-143-3p, miR-155-5p, miR-16-5p, miR-195-5p, miR-21-5p, miR-214-3p, miR-22-3p, miR-24-3p, miR-26a-5p, miR-26b-5p, miR-494.
Cardiac Hypertrophy:Up-Regulated: let-7b-5p, let-7c, miR-103a-3p miR-125b-5p, miR-140-5p, miR-142-3p, miR-146a-5p, miR-18b-5p, miR-195-5p, miR-199a-5p, miR-208a, miR-208b, miR-21-5p, miR-214-3p, miR-221-3p, miR-222-3p, miR-224-5p, miR-23a-3p, miR-23b-3p, miR-24-3p, miR-25-3p, miR-27a-3p, miR-27b-3p, miR-31-5p, miR-424-5p.Down-Regulated: miR-1, miR-126-5p, miR-133a, miR-133b, miR-149-5p, miR-150-5p, miR-181b-5p, miR-185-5p, miR-29a-3p, miR-29b-3p, miR-29c-3p, miR-30e-5p, miR-451a, miR-486-5p, miR-93-5p.Regulated: miR-182-5p.
Cardiomyopathy:Up-Regulated: let-7c, miR-100-5p, miR-103a-3p, miR-10b-5p, miR-125b-5p, miR-140-5p, miR-145-5p, miR-146a-5p, miR-181b-5p, miR-195-5p, miR-208a, miR-208b, miR-21-5p, miR-210, miR-214-3p, miR-221-3p, miR-222-3p, miR-23a-3p, miR-328, miR-342-3p, miR-423-3p, miR-499a-5p.Down-Regulated: miR-1, miR-125a-5p, miR-126-5p, miR-133a, miR-133b, miR-143-3p, miR-29b-3p, miR-365a-3p, miR-378a-3p, miR-7-5p, miR-92a-3p.
Differentiation/Development:Up-Regulated: let-7a-5p, let-7b-5p, let-7c, let-7d-5p, let-7f-5p, miR-1, miR-133a, miR-143-3p, miR-144-3p, miR-145-5p, miR-17-5p, miR-181a-5p, miR-206, miR-208a, miR-21-5p, miR-24-3p, miR-26a-5p, miR-27a-3p, miR-27b-3p, miR-30a-5p, miR-30c-5p, miR-30d-5p, miR-378a-3p, miR-93-5p, miR-98-5p, miR-99a-5p.Down-Regulated: miR-124-3p, miR-125b-5p, miR-183-5p, miR-302a-3p, miR-302b-3p.
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Technologies for total RNA discovery – lncRNA expression
qPCR arrays and assays
RT2 lncRNA PCR Arrays (link):Four pathways, available in human and mouse lncFinder Cancer PathwayFinder
Cell Development & Differentiation
Inflammatory Response & Autoimmunity
Custom lncRNA PCR arrays (link): >25,000 human lncRNA assays available
>10,000 mouse lncRNA assays
Can combine lncRNA with mRNA assays
Individual lncRNA qPCR assays (link)
Designed against combined NCBI RefSeq and Ensembl GENCODE database
Compatible with RT2 First Strand Kit and RT2 SYBR Green Mastermix
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Cardiovascular Diseases Research Portal
Visit the research portal here
Links to 24 ebiology stores on CVD-related topics, such as: Angiogenesis
Apoptosis
ECM and adhesion molecules
Hypertension
And many more
Includes tools for: mRNA profiling
microRNA profiling
lncRNA profiling
NGS
Reporter assays
Methylation arrays
RNAi
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Cardiovascular system pathway maps at GeneGlobe
500+ downloadable, editable pathway maps
Multiple maps related to cardiovascular system and diseases:
Cardiomyocyte differentiation Factors promoting cardiogenesis Angiopoietin-TIE2 signaling C. pneumoniae infection in
atherosclerosis Embryonic cell differentiation into
cardiac lineage NFAT in cardiac hypertrophy VEGF pathway Many more
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Questions?
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Ali Bierly, Ph.D.